Viruses provide a novel and powerful paradigm for the experimental examination of eukaryotic posttranslational regulatory mechanisms. Unique amongst the DNA viruses, the poxviruses complete their replicative cycle entirely within the cytoplasmic compartment of infected cells. Virion assembly is complicated by the fact that poxviruses produce several different infectious forms (IMV, intracellular mature virus; IEV, intracellular enveloped virus; CEV, cell-associated enveloped virus; and EEV, extracellular enveloped virus) all of which may play distinct roles in vivo. The structure and protein composition of the poxvirus virion also appears to dictate whether it is able to productively interact with the cytoskeleton of the infected cell in order to effect it's propulsion by polymerizing actin rockets and subsequent egress from the infected cell. Thus, in order to productively replicate, poxviruses must be able to -express, activate, localize and concentrate their numerous (>250) encoded gene products at the correct intracellular sites to support viral transcription, replication of the viral genome and the assembly of infectious progeny virions. To help direct viral protein traffic, poxviruses such as vaccinia virus (VV) have adopted many of the same protein modification, activation and targeting pathways used by their cellular hosts including glycosylation, phosphorylation, proteolytic processing and, in particular, acylation. Remarkably, our laboratory has discovered that VV encodes at least a dozen acylproteins modified either by palmitylation or myristylation. The previous grant period was primarily spent completing an identification of the palmitylated VV gene products and demonstrating that these proteins require this posttranslational modification in order to functionally participate in acquisition of the IEV envelope. In the experiments detailed for the upcoming grant period, we propose to shift our focus to the VV myristylproteins. Myristylation is a particularly interesting modification as it can occur in two different forms (typical N-terminal myristylation and atypical internal myristylation) conferring a number of different phenotypic properties to the modified protein, including membrane affinity, protein multimerization, enzyme activity, and a reversible regulatory switch. A detailed structure-function analysis of the VV myristylproteins encoded by the L1R, Al 6L, G9R, E7R and A25L genes will be undertaken with the dual goals of (i) understanding the biochemistry and functional significance of the myristyl modification, and (ii) elucidation of the biological function of the encoded gene product and what role it plays in the viral replicative cycle. The results of these experiments should extend our knowledge of the VV replicative process and enhance our understanding of the biology of acylproteins in general.